This work shows that the striking spectral changes seen upon phenol binding are due to close physical association of the flavin and phenolate. It also identifies the structural class of OYE and suggests that if NADPH is its true substrate, then OYE has adopted NADPH dependence during evolution.

I-TevI is a site-speci®c, sequence-tolerant intron endonuclease. The crystal structure of the DNA-binding domain of I-TevI complexed with the 20 bp primary binding region of its DNA target reveals an unusually extended structure composed of three subdomains: a Zn ®nger, an elongated segment containing a minor groove-binding a-helix, and a helix±turn±helix. The protein wraps around the DNA, mostly following the minor groove, contacting the phosphate backbone along the full length of the duplex. Surprisingly, while the minor groove-binding helix and the helix±turn± helix subdomain make hydrophobic contacts, the few base-speci®c hydrogen bonds occur in segments that lack secondary structure and¯ank the intron insertion site. The multiple base-speci®c interactions over a long segment of the substrate are consistent with the observed high site speci®city in spite of sequence tolerance, while the modular composition of the domain is pertinent to the evolution of homing endonucleases. Keywords: crystal structure/endonuclease/ helix±turn±helix/minor groove/Zn ®nger Introduction Intron-encoded endonucleases are proteins that promote the ®rst step in the mobility of the intron at the DNA level (Belfort and Roberts, 1997). They recognize and cleave an intronless allele of their cognate gene, initiating a replicative gene conversion event that results in the recipient allele also becoming intron-plus. These enzymes are therefore termed homing endonucleases and are grouped into a number of families based on the presence of conserved sequence elements. These are the LAGLIDADG, GIY-YIG, H-N-H and His-Cys box families (Belfort et al., 2001).I-TevI, the group I intron-encoded endonuclease of the td gene of bacteriophage T4, is the best studied member of the GIY-YIG family (Kowalski et al., 1999). The 28 kDa enzyme speci®cally recognizes its lengthy DNA substrate, or homing site, as a monomer ( Figure 1A; Mueller et al., 1995), exhibiting a high degree of sequence tolerance (Bryk et al., 1993). No single nucleotide in the 37 bp target is essential for binding and cleavage, and many multiple substitutions are well tolerated (Bryk et al., 1993. Consistent with this sequence tolerance, ethylation and methylation interference studies indicated that most of the protein±DNA contacts are via the minor groove and the phosphate backbone ( Figure 1B) (Bryk et al., 1993). The primary binding region of the enzyme is~20 bp in length, spanning the intron insertion site (IS), with a second region of contact close to the cleavage site (CS), 23±25 bp upstream of the IS ( Figure 1A). I-TevI demonstrates remarkable¯exibility, recognizing and cleaving homing site derivatives with large deletions (up to 16 bp) and insertions (up to 5 bp) between the IS and CS .The two-domain nature of the homing site is mirrored by the structure of the enzyme ( Figure 1A). I-TevI consists of two functionally distinct domains: an N-terminal catalytic domain and a C-terminal DNA-binding domain, separated by a long¯exible linker (Derbyshire et al., 1997). The catalytic ...

Over the past two years, through an NSF RCN UBE grant, the ASBMB has held regional workshops for faculty members and science educators from around the country that focused on identifying: 1) core principles of biochemistry and molecular biology, 2) essential concepts and underlying theories from physics, chemistry, and mathematics, and 3) foundational skills that undergraduate majors in biochemistry and molecular biology must understand to complete their major coursework. Using information gained from these workshops, as well as from the ASBMB accreditation working group and the NSF Vision and Change report, the Core Concepts working group has developed a consensus list of learning outcomes and objectives based on five foundational concepts (evolution, matter and energy transformation, homeostasis, information flow, and macromolecular structure and function) that represent the expected conceptual knowledge base for undergraduate degrees in biochemistry and molecular biology. This consensus will aid biochemistry and molecular biology educators in the development of assessment tools for the new ASBMB recommended curriculum.

The crystal structure of a betaThr26Ala mutant of human follicle-stimulating hormone (hFSH) has been determined to 3.0 A resolution. The hFSH mutant was expressed in baculovirus-infected Hi5 insect cells and purified by affinity chromatography, using a betahFSH-specific monoclonal antibody. The betaThr26Ala mutation results in elimination of the betaAsn24 glycosylation site, yielding protein more suitable for crystallization without affecting the receptor binding and signal transduction activity of the glycohormone. The crystal structure has two independent hFSH molecules in the asymmetric unit and a solvent content of about 80%. The alpha- and betasubunits of hFSH have similar folds, consisting of central cystine-knot motifs from which three beta-hairpins extend. The two subunits associate very tightly in a head-to-tail arrangement, forming an elongated, slightly curved structure, similar to that of human chorionic gonadotropin (hCG). The hFSH heterodimers differ only in the conformations of the amino and carboxy termini and the second loop of the beta-subunit (L2beta). Detailed comparison of the structures of hFSH and hCG reveals several differences in the beta-subunits that may be important with respect to receptor binding specificity or signal transduction. These differences include conformational changes and/or differential distributions of polar or charged residues in loops L3beta (hFSH residues 62-73), the cystine noose, or determinant loop (residues 87-94), and the carboxy-terminal loop (residues 94-104). An additional interesting feature of the hFSH structure is an extensive hydrophobic patch in the area formed by loops alphaL1, alphaL3, and betaL2. Glycosylation at alphaAsn52 is well known to be required for full signal transduction activity and heterodimer stability. The structure reveals an intersubunit hydrogen bonding interaction between this carbohydrate and betaTyr58, an indication of a mechanism by which the carbohydrate may stabilize the heterodimer.

The past 5 years have seen tremendous progress in our knowledge of old yellow enzyme (OYE) as a number of OYEs have been cloned and expressed, a high-resolution crystal structure has been determined for one of these, and new substrates have been found that can be turned over by the enzyme. Together these studies do not yet define the physiological role of OYE, but they lead to significant new insights into the enzymatic properties and structure-function relations of OYE.